For plants, glutathione conjugation is a major pathway to detoxify organic xenobiotic. Glutathione S-conjugates (SG-conjugates) are formed in the cytosol, the in vitro transport over the tonoplast has been described and a final storage in the vacuole has been postulated. We show here that alachlor rapidly accumulates as GS-conjugates in the plant vacuole and that the first step of its degradation, the formation of the respective gamma-glutamylcysteinyl-S-conjugate, is catalyzed by a vacuolar carboxypeptidase, These results suggest the glutathione conjugate as a transport form but not a storage form of xenobiotic molecules.